Article Artificial Feeding of All Consecutive Life Stages of Ixodes ricinus Nina Militzer 1, Alexander Bartel 2 , Peter-Henning Clausen 1, Peggy Hoffmann-Köhler 1 and Ard M. Nijhof 1,* 1 Institute of Parasitology and Tropical Veterinary Medicine, Freie Universität Berlin, 14163 Berlin, Germany; [email protected] (N.M.); [email protected] (P.-H.C.); [email protected] (P.H.-K.) 2 Institute for Veterinary Epidemiology and Biostatistics, Freie Universität Berlin, 14163 Berlin, Germany; [email protected] * Correspondence: [email protected]; Tel.: +49-30-838-62326 Abstract: The hard tick Ixodes ricinus is an obligate hematophagous arthropod and the main vector for several zoonotic diseases. The life cycle of this three-host tick species was completed for the first time in vitro by feeding all consecutive life stages using an artificial tick feeding system (ATFS) on heparinized bovine blood supplemented with glucose, adenosine triphosphate, and gentamicin. Relevant physiological parameters were compared to ticks fed on cattle (in vivo). All in vitro feedings lasted significantly longer and the mean engorgement weight of F0 adults and F1 larvae and nymphs was significantly lower compared to ticks fed in vivo. The proportions of engorged ticks were significantly lower for in vitro fed adults and nymphs as well, but higher for in vitro fed larvae. F1-females fed on blood supplemented with vitamin B had a higher detachment proportion and engorgement weight compared to F1-females fed on blood without vitamin B, suggesting that vitamin B supplementation is essential in the artificial feeding of I. ricinus ticks previously exposed to gentamicin. Citation: Militzer, N.; Bartel, A.; Keywords: Ixodes ricinus; artificial tick feeding; in vitro tick feeding; vitamin B; life cycle Clausen, P.-H.; Hoffmann-Köhler, P.; Nijhof, A.M. Artificial Feeding of All Consecutive Life Stages of Ixodes ricinus. Vaccines 2021, 9, 385. 1. Introduction https://doi.org/10.3390/vaccines 9040385 Ticks are obligate hematophagous arthropods and divided in three families: hard ticks (Ixodidae), soft ticks (Argasidae), and the monotypic Nuttalliellidae [1–3]. About ten Academic Editor: William C. Wilson percent of the approximately 900 known tick species are of medical or veterinary relevance and may cause direct damage due to their blood feeding habit, as well as indirect damage Received: 16 March 2021 by acting as vectors for pathogens, including viruses, bacteria, and protozoan parasites [4,5]. Accepted: 10 April 2021 In the northern hemisphere, four tick species belonging to the Ixodes ricinus species complex: Published: 14 April 2021 I. ricinus, I. scapularis, I. pacificus, and I. persulcatus are of particular relevance as they may act as vectors for a number of zoonotic pathogens, including Borrelia burgdorferi sensu lato. In Publisher’s Note: MDPI stays neutral Europe, I. ricinus is widely distributed and can serve as a vector for other human pathogens, with regard to jurisdictional claims in such as tick-borne encephalitis virus, Babesia divergens, and Anaplasma phagocytophilum as published maps and institutional affil- well [6,7]. Ixodes ricinus is a three-host tick species; all life stages (larvae, nymphs and iations. adult females) require a blood meal from different hosts for their development. This tick species also has an extraordinary broad host range on which it can feed, ranging from small mammals to livestock, birds, reptiles and humans [8,9]. To facilitate research on hematophagous arthropods, such as mosquitoes, flies, and Copyright: © 2021 by the authors. ticks, in vitro feeding techniques have found wide application [10–14]. In addition, they Licensee MDPI, Basel, Switzerland. also contribute to the 3R principle to reduce, replace, and refine the use of animals in This article is an open access article research. Artificial tick feeding systems (ATFS) have also found increased use in recent distributed under the terms and years to study tick biology, tick-pathogen interactions, drug development, and development conditions of the Creative Commons of anti-tick vaccines under defined laboratory conditions [15–20]. ATFS also found a wide Attribution (CC BY) license (https:// application for identifying different tick control targets or the development of anti-tick creativecommons.org/licenses/by/ vaccines under defined laboratory conditions [17–20]. 4.0/). Vaccines 2021, 9, 385. https://doi.org/10.3390/vaccines9040385 https://www.mdpi.com/journal/vaccines Vaccines 2021, 9, 385 2 of 16 Feeding systems for hematophagous arthropods typically consists of four parts: (1) a unit containing the arthropods, (2) the blood meal, (3) a membrane that mimics skin and separates the arthropods from the blood meal, and 4) a temperature control system to heat the blood meal to a temperature corresponding to the body temperature of their homeothermic hosts [21–23]. In contrast to soft ticks and other hematophagous arthropods, which generally feed for short periods only, hard ticks feed for prolonged periods of up to several days or weeks; I. ricinus juvenile ticks typically feed for 3–5 days and adults for 7–12 days [24,25]. This long duration forms a major challenge in the artificial feeding process [26], as it, in combination with temperatures of approximately 37 ◦C, results in a higher risk of decay of the blood meal. This results in the need for regular blood changes, making the artificial feeding a laborious process, and the addition of antibiotics in the blood meal, which may affect the tick microbiome including nutritive symbionts [12,27–30]. In addition, hard ticks also have an intricate pre-feeding behavior [24,26], the mimicking of which in vitro can be complicated. Hard ticks including I. ricinus are commonly reared on experimental animals [31], although reports on the in vitro feeding of nymph and adult I. ricinus have been pub- lished [15,32–34]. The only hard tick species for which successful feeding of all life stages has previously been reported is the tropical bont tick Amblyomma hebraeum [27]. Here, we report on the completion of the life cycle of I. ricinus in vitro by the feeding of all consecu- tive life stages using an ATFS and a comparison between relevant biological parameters of in vitro fed ticks and ticks fed on cattle, further referred to as in vivo fed ticks. 2. Materials and Methods 2.1. Tick Feeding All ticks used for this study originated from a laboratory colony at the Institute of Parasitology and Tropical Veterinary Medicine of the Freie Universität Berlin. For the maintenance of this colony, larvae are routinely fed on laboratory gerbils (Meriones unguiculatus), nymphs on rabbits (Oryctolagus cuniculus), and adults on rabbits or calves (Bos taurus). All replete larvae and nymphs are kept at room temperature and >90% Relative Humidity (RH). Shortly after molting into the adult stage, ticks are separated by sex and stored at 12 ◦C and >90% RH in the dark. Replete adult ticks are kept in the dark at 20 ◦C, >90% RH. For this study, 8 to 32-week-old and 8 to 15-week-old larvae were used for the in vivo and in vitro feeding, respectively. In vivo fed nymphs were fed at an age of approximately 2–4 months post molt, in vitro nymphs at 2–3 months. In vitro F0-adult ticks were fed at an age of 7–9 months post molt, while in vitro F1-adult ticks were fed at 5–7 months. Adults fed in vivo had an age range of 2–10 months post molt. For our study, all life stages reported as in vivo fed ticks were fed inside linen ear bags on 14 to 18-week-old tick-naïve Holstein-Friesian calves. The ears were checked twice daily for engorgement starting at three or five days post-infestation for juvenile and adult ticks, respectively. Thirty female and 30 male I. ricinus adults, nine months after being fed on rabbits, were used to initiate the in vitro life cycle (F0 adults). They were brought together in a desiccator kept at room temperature and >90% RH seven days before the start of the artificial feeding. Blood used for in vitro feeding was drawn aseptically from cattle that grazed on pastures considered to be free of ticks; natural tick infestations were not observed during this period. All animal experiments were approved by the regional authorities for animal experiments (Landesamt für Gesundheit und Soziales, Berlin, Germany, 0387/17). 2.2. Artificial Tick Feeding System (ATFS) The ATFS developed by Kröber and Guerin [1] was adapted as previously reported [35]. For containment of the blood, autoclaved 50 mL beaker glasses (SIMAXX, Bohemia Cristal, Selb, Germany) or sterile standard 6-well cell culture plates were used. Ten females and Vaccines 2021, 9, 385 3 of 16 10 males were placed in each feeding unit. Juvenile ticks were fed using a smaller feeding system, which fitted in a standard 12-well cell culture plate (Sarstedt, Nürnbrecht, Ger- many). Here, the feeding units were made up of a borosilicate glass tube (length 40 mm, inner diameter 15–16 mm; Neubert Glass Geschwenda, Germany and Glastechnik Rahm Mutterz GmbH, Switzerland), and a smaller rubber ring with an inner diameter of 18 mm (Emil Lux GmbH, Wermelskirchen, Germany). A moistened air-permeable foam plug (K-TK e.K., Retzstadt, Germany) was used instead of a plastic stopper for the smaller feeding units. 2.3. Artificial Membranes The silicone mixture for the artificial membranes was produced as previously de- scribed [32,35]. A metal scraper (Emil Lux) was used to spread the silicone paste onto a matrix made of lens cleaning paper for adult ticks (Tiffen, Happauge, NY, USA) or goldbeater’s membrane for juvenile ticks (20 µm thickness, Altenburger Pergament and Trommelfell GmbH, Altenburg, Germany). After overnight drying, the membranes were glued to glass tubes using silicone glue (Elastosil E41, Wacker, München, Germany).